The objective of this proposal is to study a newly identified system that regulates the contractile properties of the myofibrils in mammalian cardiac cells with particular emphasis on the molecular mechanism and the normal physiological function in the intact organ and organism. This regulatory system is under the control of the adrenergic system, and preliminary data indicate that it is sensitive to the mechanical and metabolic state of the heart. The active component appears to be a 20Kd protein, which may be the phosphorylatable light chain of myosin. The working hypothesis is that myosin light chains are not tightly bound to the heavy chain at normal body temperature and normal resting cytosolic calcium concentration, and a second binding site for the 20Kd light chain exists on a 15Kd protein in a cell membrane. Beta adrenergic stimulation causes a phosphorylation of the 15Kd protein that lowers its affinity for the 20Kd protein and increases cytosolic calcium, both of which enhance binding of the 20Kd protein to myosin in the thick filament. Binding of the 20Kd protein to myosin increases its ability to function as a force generator. Cryostatic sections of hearts that have been frozen quickly will be exposed to solutions with different concentrations of Ca, ATP, pH at different temperatures and with different concentrations of beta adrenergic agonists. ATPase activity of myosin will then be measured by a microphotometrical procedure based on trapping the phosphate liberated by ATP hydrolysis in the tissue section. Myosin will then be extracted, and analyzed by gel electrophoresis to determine the amount of 20Kd protein amount still present in the tissue. Correlations with ATPase activity will be examined. Antibodies will be used to confirm or refute the LC2 identify of the 20Kd protein. The role of this system in the response of the heart to changes in mechanic and metabolism will be examined with isolated working hearts. Labelled antibodies will also be used for localization of 20Kd protein in the myocardial cells. This work sould provide greater understanding of a regulatory system that can change the contractile activity of the heart over a 7 fold range; it will allow more rational regemins for managing cardiac diseases pharmacologically as well as adding to understanding of factors influencing the heart during surgery, in particular during bypass and/or cooling.